Bacillus thuringiensis is a gram positive bacterium that produces a variety of crystalline protein toxins during sporulation generally referred to as delta-endotoxins or Cry proteins. Many of these are highly toxic to a range of agronomic insect pests but are generally harmless to mammals and most other organisms. One such delta-endotoxin, Cry1Ca, is insecticidal to certain lepidopteran pests found in North and South America corn fields.
Cry1Ca has been shown to effectively control fall armyworm, Spodoptera frugiperda, and Cry1Fa resistant fall armyworm when expressed in maize plants as a full length protein (Sheets, J., et al., Entomological Society of America, Annual Meeting, Nov. 12, 2013, Austin Tex.). The full length Cry1Ca holotoxin is cleaved by native enzymes in the insect gut to produce a core toxin having approximately 624 residues of the amino terminus depending on the insect and gut conditions. Cry1Ca core toxin-containing proteins and genes are therefore attractive candidates for developing recombinant crop plants such as corn, soy, cotton, canola, and others often referred to as genetically modified (GM) plants.
Companies which develop and market GM crop seeds containing recombinant DNA that confer beneficial new traits are required to formulate, implement and adhere to strict product stewardship plans. These stewardship plans require the use of validated quantitative and qualitative protein detection methods for the recombinant protein to track trait introgression and seed production activities, as well as to monitor the GM trait during and after harvest. These detection methods must be facile and robust enough to use under good laboratory practice (GLP) and non-GLP conditions. Moreover the methods must be user friendly enough to be easily employed by farmers in the field, grain dealers at the silo, and customs officials at the borders. Therefore, robust, high quality, user friendly protein detection methods and commercial kits are useful and necessary.
While immunoassays are well-known in the art, developing robust, high quality, validated ELISA methods that are reproducibly able to detect a particular transgenic protein product in an array of plant tissue in both lab and field settings is neither trivial nor routine. Still more challenging is to find antibody pairs that are particularly well suited to the development of a lateral flow strip ELISA for detecting Cry1Ca expressed by a transgenic event in a crop.